Peptide purification

ABSTRACT

A nona- or decapeptide is purified from residual organic solvent by dissolving in a solvent comprising water and at least one C 1 -C 3  alcohol followed by precipitation into a vigorously stirred solvent consisting of an alkyl ester of a carboxylic acid, the ester comprising from  3  to  6  carbon atoms, and one or several non-polar compounds selected from hexane, heptane, octane, cyclohexane, methylcyclohexane, and, optionally, of up to  5 % of acetic or propionic acid, isolating the precipitated nona- or decapeptide, followed by washing with a mixture of C 3 -C 5  esters and drying, with the provisio that the water content of the solvent comprising water and the at least one alcohol is below  8 % (v/v), and that the volume ratio of the dissolution solvent mixture and the precipitation solvent mixture is  1:10  or higher. Also described is the monoacetate of Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-MeTyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH 2 .

FIELD OF THE INVENTION

The present invention relates to the purification of peptides, inparticular intermediate-size peptides, more particularly nona- anddecapeptides, such as LHRH-antagonists

BACKGROUND OF THE INVENTION

Most intermediate size natural and synthetic peptides are amorphoussubstances. Many of them have pharmacologically. interesting properties,such as many nona- and decapeptides which are LHRH (luteinizinghormone-releasing hormone) antagonists. One particular substance of thiskind known only in amorphous form is the synthetic decapeptide of theformula (I)Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-MeTyr-D-Asn-Leu-Lye(iPr)-Pro-D-Ala-NH₂  (I)which, being a potent LHRH antagonist, has desirable pharmacologicalproperties.

For use in pharmaceutical preparations it is necessary for the LHRHantagonist (I) and nona- and decapeptides of similar structure to beessentially pure. The raw product obtained in the last step of amultiple-step synthesis is purified by chromatographic and othermethods. To eliminate residual solvent from the chromatography a thuspurified product usually has to be dissolved in an aqueous medium andfreeze-dried. This is a costly process producing a voluminous productwhich is not easy to handle.

A process of purification of an otherwise pure peptide from residualorganic solvent by other means than freeze-drying thus is desirable.

Wo 0018423 A discloses the use of the combination of cyclodextrin and aLHBR peptide analogue, including its pharmaceutically acceptable salts,for the preparation of a pharmaceutical composition for oraladministration. Abarelix is a preferred LHRM analogue. Acetate salts ofthe LHRH peptide antagoniste of D1 are disclosed to be pharmaceuticallyacceptable in general. The compounds of WO 001842.3 are useful in themanufacture of medicaments.

WO 0055190 A discloses decapeptide LHRM antagonists for the manufactureof medicaments against hormone dependent tumors and hormone influenceddiseases, which may be purified by preparative HPLC, and freeze dried.

EP 095530 A discloses a process for the transformation or hydrochloridesalts of decapeptide LHRH antagonists to their diacetate salts in aconcurrent single-step purification by liquid chromatography.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide a process ofpurification of an otherwise pure peptide of the aforementioned kindfrom residual organic solvent which avoids freeze-drying.

It is another object of the present invention to provide such anotherwise pure peptide which is essentially free from residual organicsolvent and is not in the form of a cryoprecipitate.

Further objects of the invention will become obvious from the followingsummary of the invention, the description of a preferred embodimentthereof, And the appended claims.

SUMMAY OF THE INVENTION

According to the present invention is provided a process of purificationof an otherwise pure peptide, in particular a nona- or decapeptide, mostparticularly a nona- or decapeptide which is an LHRH antagonist, fromresidual organic solvent, comprising the following steps:

-   -   dissolving said otherwise pure peptide in a dissolution solvent        mixture comprising water and at least one alcohol selected from        methanol, ethanol, propanol, isopropanol;    -   adding the solution of the otherwise pure peptide in said        solvent mixture to a vigorously stirred precipitation solvent        mixture essentially consisting of one or several polar compounds        selected from methyl acetate, ethyl acetate, methyl propionate,        ethyl propionate, butyl acetate, isobutyl acetate, t-butyl        acetate, ethyl formate, propyl formate, isopropyl formate, and        one or several non-polar compounds selected from hexane,        heptane, octane. cyclohexane, methylcyclohexane, and,        optionally, of up to 5% of acetic or propionic acid;    -   isolating the precipitated peptide;    -   washing the isolated peptide with one or a mixture of said polar        compounds or a solvent or solvent mixture of said polarity,    -   drying the washed peptide, with the provisio that the water        content of said solvent mixture comprising water and at least        one alcohol is below 8% (v/v), and that the volume ratio of the        dissolution solvent mixture and the precipitation solvent        mixture is 1:10 or more. “An otherwise pure peptide” is a        peptide which is sufficiently pure for use in a medicine except        for volatile impurities which need to be removed or the content        of which needs to be substantially reduced. The otherwise pure        peptide will normally be a substance having undergone        purification by chromatography.

Preferably the otherwise pure peptide isAc-D-2Nal-D-4ClPhe-D-3Pal-Ser-MeTyr-D-Asn-Leu-Lye(iPr)-Pro-D-Ala-NH₂  (I).

According to a first preferred aspect of the invention the water contentof the dissolution solvent mixture is below 5% (v/v).

According to a second preferred aspect of the invention the volume ratioof the dissolution solvent mixture and the precipitation solvent mixtureis at least 15, in particular at least 20.

According to a third preferred aspect of the invention the alcohol ofthe dissolution solvent mixture is ethanol.

According to a fourth preferred aspect of the invention the polarcomponent of the precipitation solvent mixture is ethyl acetate.

According to a fifth preferred aspect of the invention the non-polarcomponent of the precipitation solvent mixture is heptane.

In the following the invention will be described in greater detail byreference to a preferred embodiment thereof which should not beunderstood to limit the invention.

DESCRIPTION OF A PREFERRED EMBODIMENT EXAMPLE 1

The fractions containing pure compound (1) (100 g in total) inethanol-water-acetic acid 40:59:1 (v/v/v) obtained from preparative HPLCby which the synthetically obtained product had been purified werepooled and concentrated in vacuo to an oil which was co-evaporated twicefrom ethanol. The resulting solid was dissolved in 440 ml of ethanol andthe resulting clear solution added over a period of 20 min to 8.8 L ofethyl acetate/heptane 1:1 (v/v).

Stirring was continued for 1 hour and filtered. The amorphous productwas washed with 3 L of ethyl acetate; it was found that this removednearly all heptane. The washed product was dried in a vacuum oven at 40°C. at 0.3 bar for 48 hrs. Elemental analysis of the dried productindicated that the monoacetate of (I) had be obtained.Cryoprecipitation, in contrast, produces the corresponding diacetate. Inthe following Table analytical parameters of the monoacetate of (I)produced according to the invention are compared with those of acorresponding lyophilized product (diacetate). TABLE All percentages areby weight lyophilized product precipitated product Water 3.4% 3.0%Ethanol <0.024% <0.024% Ethyl acetate <0.024% <0.024% Heptane <0.024%<0.024% Acetic acid (as acetate) 7.5% 3.9% HPLC purity 99.8% 99.8%Peptide content 88.8% 94.9% Loss in filtrate — 0.3%

EXAMPLE 2

Variation of the composition of the precipitation solvent or solventmixture: otherwise, procedure as in Example 1. Precipitation in pureethyl acetate results in from about 3% to about 5% by weight loss ofpeptide. Precipitation in pure heptane results in a sticky product whichis difficult to filter. A 1:1 (v/v) mixture of ethyl-acetate hexanelives a product which is easy to filter and dry; in repeated experimentsthe loss of peptide was always leas than 0.5% by weight.

EXAMPLE 3

variation of water content of the solution of oily product in thedissolution solvent (absolute ethanol), otherwise, procedure as inExample 1. A water content of 10% (v/v) results in a sticky product onprecipitation which is difficult to filter. A water content of 15% (v/v)results in an oily product on precipitation. To obtain satisfactoryresult the water content must not exceed 8% (v/v) but should preferablybe kept below 5% (v/v). A water content below 5% is accomplished byco-evaporating the oily product from the chromatography at least twicewith ethanol.

EXAMPLE 4

Variation in precipitation temperature: otherwise, procedure as inExample 1. The precipitation temperature proved to be not critical. Itcould be varied from 0° C. to 20° C. without noticeable differences inproduct yield and morphology.

EXAMPLE 5

Variation of concentration of (I) in the dissolution solvent; otherwise,procedure as in Example 1. It was found that the concentration of theoily product from the chromatography which had been co-evaporated withethanol in the dissolution solvent should be as high as possible. Even aconcentration of 330 g by weight could be used.

EXAMPLE 6

Variation of ratio between dissolution solvent and precipitation solventvolumes and other variations otherwise, procedure as in Example 1. Theoptimum ratio was found to be about 1:20. It could be shown that ratiosfrom 1:15 to 1:30 gave satisfactory results. A ratio of 1:10 resulted ina sticky product. Precipitation is very fast. The suspension can befiltered 30 min after the last addition of dissolved substance. Washingwith ethyl acetate did not result in loss of product but efficientlyremoved heptane.

1. A process of purification of an otherwise pure nona- or decapeptidefrom residual organic solvent, comprising the following steps:dissolving the nona- or decapeptide in a dissolution solvent mixturecomprising water and at least one alcohol selected from methanol,ethanol, propanol, isopropanol; adding the solution of the nona- ordecapeptide in said solvent mixture to a vigorously stirredprecipitation solvent mixture essentially consisting of one or severalpolar compounds selected from methyl acetate, ethyl acetate, methylpropionate, ethyl propionate, butyl acetate, isobutyl acetate, t-butylacetate, ethyl formate, propyl formate, isopropyl formate, and one orseveral non-polar compounds selected from hexane, heptane, octane,cyclohexane, methylcyclohexane, and, optionally, of up to 5% of aceticor propionic acid; isolating the precipitated nona- or decapeptide;washing the isolated nona- or decapeptide with one or a mixture of saidpolar compounds or a solvent or solvent mixture of similar polarity,drying the washed nona- or decapeptide, with the proviso that the watercontent of said solvent mixture comprising water and at least onealcohol is below 8% (v/v), and that the volume ratio of the dissolutionsolvent mixture and the precipitation solvent mixture is 1:10 or more.2. The process of claim 1, wherein the water content of said solventmixture comprising water and at least one alcohol is below 5% (v/v). 3.The process of claim 1, wherein said nona- or decapeptide is an LHRHantagonist.
 4. The process of claim 3, wherein said nona- or decapeptideis Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-MeTyr-D-Asn-Leu-Lye(iPr)-Pro-D-Ala-NH₂  (I).
 5. The process of claim 4, whereinAc-D-2Nal-D-4ClPhe-D-3Pal-Ser-MeTyr-D-Asn-Leu-Lye (iPr)-Pro-D-Ala-NH₂  (I) is obtained in form of the monoacetate.
 6. The process of claim 1,wherein the volume ratio of the dissolution solvent mixture and theprecipitation solvent mixture is at least
 15. 7. The process of claim 1,wherein the alcohol of the dissolution solvent mixture is ethanol. 8.The process of claim 1, wherein the polar component of the precipitationsolvent mixture is ethyl acetate.
 9. The process of claim 1, wherein thenon-polar component of the precipitation solvent mixture is heptane. 10.The monoacetate ofAc-D-2Nal-D-4ClPhe-D-3Pal-Ser-MeTyr-D-Asn-Leu-Lye(iPr)-Pro-D-Ala-NH₂.